Method for the detection and classification of PRRSV-infections in swine herds and diagnostic antigen compositions for such methods

ABSTRACT

Method for the detection and classification of PRRSV-infections in swine herds, comprising a) the incubation of tissue samples taken from the animals with at least one antigen capable to bind a neutralizing antibody against the Type I-virus possibly present in the animal and with at least one antigen capable to bind a neutralizing antibody against the Type II-virus possibly present in the animal, b) testing whether a binding of antibodies against the Type I-virus and/or the Type II-virus has taken place and c) determining from the presence of possible epitope-antibody complexes whether an infection of the PRRSV I-Type and/or PRRSV II-Type is present in the herd and diagnostic compositions for such a method.

CROSS REFERENCE TO PRIORITY APPLICATIONS

This Application is a divisional of U.S. application Ser. No. 14/890,643filed Nov. 12, 2015, which is a U.S. National Phase Stage under § 371 ofPCT/EP2014/001301 filed May 14, 2014, which claims priority toEP13002540.6 filed May 15, 2013. The entire contents of theaforementioned applications are incorporated by reference herein.

SEQUENCE LISTINGS

This application contains a nucleotide and/or amino acid SequenceListing which has been submitted as LT00998US_ST25.txt, via EFS-Web andis hereby incorporated by reference in its entirety.

The invention relates to a method according to claim 1 and diagnosticcompositions according to claim 9.

Porcine reproductive and respiratory syndrome (PRRS) is one of the mostdevastating viral diseases causing significant economic losses to theswine industry. The agent responsible for this endemic disease is thePRRS virus (PRRSV) which is easily transmitted via infected pigs throughurine, semen and fomites, infecting almost exclusively pig monocytes andmacrophages.

Porcine reproductive and respiratory syndrome (PRRS) emerged first inthe United States of America in 1987 and subsequently spread worldwide.The syndrome is characterized mainly by reproductive failure in pregnantsows, high mortality in piglets and respiratory problems such asdyspnoea. The cause of these symptoms is an enveloped positivesingle-stranded RNA virus. The PRRSV belongs to the Arteriviridae familyand it infects almost exclusively swine monocytes and macrophages.

The genome is approximately 15 kb and has an UTR region on 5′ followedby nine open reading frames (ORF): ORF1a, ORF1b, ORFs2a and 2b, 3 to 7.ORF1a and ORF1b constitute about 75% of the virus genome encoding forproteins with replicase and polymerase activities, whereas ORF 2 to 5appear to encode for membrane-associated proteins or glycoproteins (GP).ORF6 encodes for a viral unglycosylated membrane protein M and ORF7encodes for a highly conserved basic protein, N, that constitutes thenucleocapsid. Mutations take place predominantly in the nsp2 region ofORF 1a and the envelope proteins GP3, GP4 and GP5.

Both Type I and Type II strains have a number of neutralizing andimmunodominant epitopes.

Sensitivity to antibody-mediated neutralization was tested for aselected number of antigen regions (AR) by in vitro virus-neutralizationtests on alveolar macrophages with peptide-purified antibodies. Inaddition to the known neutralizing epitope in GP4, one in GP3 turned outto be targets for virus neutralizing antibodies. Since the neutralizingAR in GP3 induced antibodies in a majority of infected pigs, theimmunogenicity of this AR was studied extensively, and it wasdemonstrated that the corresponding region in GP3 of virus strains otherthan Lelystad Virus (PRRSV type 1) also induces virus neutralizingantibodies. This provides new insights into PRRSV antigenicity, andcontributes to the knowledge on protective immunity and immune evasionstrategies of the virus (vanhee et al; 2011).

Since ARs GP3 and GP4 were both immunogenic and target for neutralizingantibodies, the serum antibody response against these ARs was furtherstudied (vanhee et al; 2011). While antibodies against GP4 appearedrelatively fast upon infection in a majority of the animals, it clearlylasted longer for most animals to develop antibodies against AR GP3.Moreover, antibody titers in sera and alveolar fluids against GP3 wereinvariably lower than against GP4.

Based on the genetic differences of the virus, it is possible toencounter two main types: European genotype (Lelystad virus or Type I)and American genotype (VR-2332 virus or Type II). These PRRSV genotypeshave a nucleotide identity between 55% and 70% when both genomes arecompared.

Protection against PRRSV infection is not possible and prevention ofvirus replication is not mediated by the humoral response alone.Cellular immunity is critical to prevent clinical signs of PRRS.

Nowadays, there are mainly two commercially available modified livevaccines (MLV) on the market; one is composed of an EU type (Porcilis®PRRS) and the other one of a US type PRRSV (Ingelvac® PRRS). Bothvaccines demonstrate an overall efficiency and are widely used for theprevention of PRRSV induced clinical symptoms.

PRRS MLV vaccine confers effective genotype/strain-specific protection,but provides only partial protection against genetically heterologousPRRSV. Before the emergence of atypical PRRS, it was generally assumedthat the reproductive consequences of infection with PRRSV, includingabortion, were due to the direct effect of the virus on the conceptusfollowing transplacental infection. While this is certainly true formany cases of PRRSV-induced reproductive failure, it apparently does notexplain all cases, because many litters aborted during epidemics ofatypical PRRS were found free of infection. The latter observationindicates that abortion can also be the result of a systemic reaction.The additional observation that abortions were common in herds that hadpreviously been vaccinated raised the question of whether anacute-anamnestic immune response contributed to the clinical picture. Ifso, there is a possibility that exposure to a virulent field strain ofPRRSV other than that used in the vaccine elicits a response differentthan what would have followed exposure of a naive gilt or sow to thesame strain. That is not to say vaccination is not beneficial overall,but it is possible that prevalence of one aspect of reproductivefailure, namely abortion, may be slightly higher in vaccinated herdsthan in non-vaccinated herds. (W. L. Mengeling, K. M. Lager, A. C.Vorwald The effect of porcine parvovirus and porcine reproductive andrespiratory syndrome virus on porcine reproductive performance. AnimalReproduction Science 60-61_2000. 199-210)

In such cases, sows often lack neutralizing antibodies against one typeof PRRSV. This might be why it is imperative to continuously vaccinateherds with one type of PRRS MLV vaccine since it may displace the otherPRRSV type in the herd. In case of a field infection with the counterPRRSV strain, these animals may not be properly protected; crossprotection of vaccines against PRRSV of the other type is not fullyconvincingly shown.

WO 2010/062395 discloses an assay which allows the differentiation ofserum antibodies against Type I and/or Type II PRRSV utilizing PRRSVnsp7 from both strains as antigen.

In order to improve vaccine and sanitary management in farms it might beof interest to be aware of a more detailed infectious status of a herd,e.g. to distinguish chronic infections or vaccination from freshinfections.

Such information can be crucial in the further treatment of the animals.

The object of the invention is to provide an improved test method forPRSSV and diagnostic compositions for such method.

According to the invention a method is provided for the detection andclassification of PRRSV-infections in swine herds, comprising theincubation of tissue samples taken from the animals with at least oneantigen capable to bind a neutralizing antibody against the Type I-viruspossibly present in the animal and with at least one antigen capable tobind a neutralizing antibody against the Type II-virus possibly presentin the animal. After incubation it is tested, whether a binding ofantibodies against the Type I-virus and/or the Type II-virus with theantigens has taken place and it is determined from the presence ofpossible antigen-antibody complexes whether an infection of the PRRSVI-Type and/or PRRSV II-Type is present in the herd.

The term tissue is used in a broad sense. Apart from its literal meaningit shall also encompass body fluids like blood, plasma, serum or urine.

The antigens used are selected from PRRSV Type I and Type II peptidesequences containing a neutralizing epitope.

Peptides of GP3 Type I (EU) which can be used with the invention includepeptides containing a sequence according to SEQ ID NO. 6. Examples forsuch peptides are apart from a peptide consisting of SEQ ID NO. 6peptides with sequences according to SEQ IDs NO. 1 and 5.

Peptides of GP3 Type II (US) which can be used with the inventioninclude peptides containing a sequence according to SEQ ID NO. 8.Examples for such peptides are apart from a peptide consisting of SEQ IDNO. 8 peptides with sequences according to SEQ IDs NO. 3 and 9.

Peptides of GP4 Type I (EU) which can be used with the invention includepeptides containing a sequence according to SEQ ID NO. 7. A furtherexample for such a peptide apart from a peptide consisting of SEQ ID NO7 is a peptide with a sequence according to SEQ ID NO. 2.

Peptides of GP4 Type I (US) which can be used with the invention includepeptides containing a sequence according to SEQ ID NO. 10. A furtherexample for such a peptide apart from a peptide consisting of SEQ ID NO10 is a peptide with a sequence according to SEQ ID NO. 4.

A list of the peptides mentioned above is given in the following:

Type I (EU) PRRSV SEQ ID NO: 1 (GP3 LV 30AA54):ICMPCSTSQA ARGRLEPGRS NMCRKGHDRC SEQ ID NO: 2 (GP4 LV4)DINCFRPHGV SAAQEKISFG KSSQCREA VGTP SEQ ID NO. 5 (GP3):STSQAARQRL EPGRNMWCKI GHDRCEER SEQ ID NO. 6 (GP3):: STSQAARQRL EPGRNMWSEQ ID NO. 7 (GP4):: FRPHGVSAAQ EKISFGKSS Type II (US) PRRSVSEQ ID NO: 3 (GP3 VR): VCPPCLTRQA ATEIYEPGRS LWCRIGYDRC EEDHDELGFMSEQ ID NO: 4 (GP4 VR): DISCLRHRDS ASEAIRKIPQ CRTAIGTPSEQ ID NO. 8 (GP3):: YEPGRSLWCR IGYDRCGEDD SEQ ID NO. 9 (GP3)::IYEPGRSLWC RIGYDRCGED DHDEL SEQ ID NO. 10 (GP4):: HRDSASEAIR KIPQCRTAI

The above mentioned peptides are examples. It is clear for a personskilled in the art that further peptides containing sequences accordingto SEQ IDs NO. 6, 7, 8 or 10, respectively, can have similar antigenicproperties which allow the diagnostic testing of animals and thus arealso encompassed by the invention. The length of such peptides islimited by their ability to differentiate between PRRSV Type I and II indiagnostic tests. Such ability should be given for peptides not longerthan 50 amino acids including the above mentioned sequences.

GP4 and GP3 protein is very well conserved between genotypes and it canbe used as a target for extremely specific serological detection by e.g.indirect Enzyme-Linked Immunosorbent Assay (ELISA), due to its capacityto produce neutralizing antibodies. The above mentioned epitopes havebeen proven to induce the generation of neutralizing antibodies first byvanhee et al; 2011, but previously described by Oleksiewicz M B, BotnerA, Toft P, Grubbe T, Nielsen J, Kamstrup S, et al. Emergence of porcinereproductive and respiratory syndrome virus deletion mutants:correlation with the porcine antibody response to a hypervariable sitein the ORF 3 structural glycoprotein. Virology 2000; 267: 135-40.Oleksiewicz M B, Botner A, Toft P, Normann P, Storgaard T. Epitopemapping porcine reproductive and respiratory syndrome virus by phagedisplay: the nsp2 fragment of the replicase polyprotein contains acluster of B-cell epitopes. J Virol 2001; 75: 3277-90.

Especially preferred is to use the sequences according to SEQ ID NO: 2,4 7 and 10 of GP4 which as stated above contains a neutralizing andimmunodominant epitope in its N-terminal for both Type I and Type IIstrains. Although most pigs are able to develop antibodies against bothARs, GP4 clearly shows the highest immunogenicity in the context ofinfection. There is evidence from recent studies that AR GP4 issusceptible to antibody-mediated selective pressure in vivo, and itshigh variability confirms that aminoacid changes in this region aregenerally well tolerated by the virus.

When the GP4 sequences of aminoacids of both Type I and Type II strainsare compared ([GenBank: AAA46277.1]; [GenBank: ABB18277.1]), it isobserved that the neutralizing epitope is situated within the mostvariable region of the GP4 protein. Since GP4 shows such greatvariability in both genotypes, it can be used as a very specific targetfor the detection of the virus in infected pigs.

As further specified below it is possible to e.g. develop indirect ELISAdetection method using a peptide containing a neutralizing epitope inorder to discriminate Type 1 and Type 2 PRSSV.

In a further preferred embodiment it is provided that to use a cocktailof peptides, e.g. the above mentioned SEQ ID Nos: 1-4. The use of such acocktail leads at least in some cases to a broader immune response.

As stated above the main feature of the invention is that method usesantigenic sequences which include neutralizing epitopes. By using suchantigens it is possible to detect the presence of neutralizingantibodies possibly present in the animals. However, neutralizingantibodies are only present in animals for a certain period postinfection or vaccination. In case that PPRSV is permanent present in agiven herd situation (boars) or repeated vaccination (in the followingsuch cases will be referred to as repeated infections) it may happenthat in infected animals no neutralizing antibodies are detectable.

For such cases it may be preferable if an additional standard assay forPRRSV is performed, which detects antibodies normally directed againstnon neutralizing epitopes (e.g. GP7). In an infectious or vaccinatedsituation animals with repeated infection turned positive in such ascreening test.

With this embodiment of the method a very detailed classification of thestatus of possible infections in the herd is possible especially iffurther information on the herd normally available, like e.g. date ofvaccination etc., is taken into account. If e.g. the standard test ispositive and the titre of neutralizing antibodies is negative or lowthis means that there is a chronic infection in the herd. If on theother hand the standard assay is positive and the test for neutralizingantibodies provides a high titre this means that there is a freshinfection in the herd.

Summing up the method according to the invention allows a much moredetailed evaluation of the infection status in a herd compared to knownassay formats since it considers the humoral immune response againstneutralizing epitopes in addition to the information available on theherd. On the basis of such evaluation pork producers can e.g. assess therisk of facing clinical signs of a PRRSV infection despite vaccinationand can adapt their vaccination strategy, e.g. consider treating animalswith either a commercially available Type I or Type II modified livevaccine. Additionally, the measurement of the neutralizing antibodytitre against PRRSV Type I or Type II may also give an indication of thefitness of a herd against potential PRRSV infection if neutralisation ABtitre of only one type can be detected but infected with the other one.This is the reason why a high specificity test as described belowcapable of distinguishing between Type I and Type II in one single runmay benefit the PRRSV diagnostic.

The invention is also directed to diagnostic compositions which can beused in the method according to the invention. Such compositions includeat least the GP4 sequences indicated above, but can also include furtherantigenic sequences (GP3). As a rule the sequences are immobilized, e.g.by coating on a plate or coupling to beads. The compositions furthermoreinclude all buffers and controls which normally are present in suchcompositions. A detailed composition is given in Example 1.

DESCRIPTION OF THE DRAWING FIGURES

In the following the invention shall be described further in detail bymeans of figures and examples:

FIG. 1 Illustration of OD 450-620 nm values for different PRRSV serameasured using EU-specific antigen,

FIG. 2 Illustration of OD 450-620 nm values for different PRRSV serameasured using US-specific antigen,

FIG. 3 Illustration of OD 450-620 nm values for different PRRSV serameasured using a combination of further EU-specific antigenic peptides,

FIG. 4 Illustration of OD 450-620 nm values for different PRRSV serameasured using a further combination of EU-specific antigenic peptides,

FIG. 5 Illustration of OD 450-620 nm values for different PRRSV serameasured using a combination of further US-specific antigenic peptides,

FIG. 6 Illustration of OD 450-620 nm values for different PRRSV serameasured using a further combination of US-specific antigenic peptides.

FIG. 1 illustrates OD 450-620 nm values for PRRS negative (CH Herds 1 to4) and PRRSV positive sera samples (Porcilis® PRRS and Ingelvac® PRRSvaccinated animals) using a high binding plate coated with 1 μg/ml ofGP4_LV4. Stabilcoat had been used for blocking and 0.2% casein inStabilcoat and PBS 13 1×+0.1% Tween-80 had been used as sample diluentand conjugate buffers, respectively.

FIG. 2 illustrates OD 450-620 nm values for PRRS negative (CH Herds 1 to3) and PRRS positive sera samples (Porcilis® PRRS and MLV or Ingelvac®PRRS vaccinated Animals) using a high binding plate coated with 10 μg/mlof GP4_VR_2332_4. Stabilcoat had been used for blocking and 0.2% caseinin Stabilcoat and 0.1% Tween-80+0.2% Casein in PBS 13 1× had been usedas sample diluent and conjugate buffers, respectively.

FIGS. 3-6 illustrate the results of tests with combinations of furtherantigenic peptides on different PRRSV sera. For details see example 11.

EXAMPLE 1: DEVELOPMENT OF AN INDIRECT ELISA

a) Antibodies

A secondary antibody was used as conjugate for the detection ofneutralizing antibodies (Goat anti Pig IgG (Fc) HRP; stock concentration1 mg/ml [AbD Serotec, AAI41P]) present in the PRRS samples.

b) Antigen

The antigen used for the development of this indirect ELISA was GP4peptide from the European PRRSV strain and the American PRRSV strainaccording to SEQ ID NOs: 1 and 2. The Type I peptide (EU) used shallalso be designated as GP4_LV4. The stock concentration of this peptidewas 3 mg/ml in H₂O. Peptide GP4_VR_2332_4 corresponds to the Type II orUS strain and had a stock concentration of 2 mg/ml in H₂O. The optimalcoating concentration of GP4_LV4 was 1 μg/ml whereas for GP4_VR_2332_4was 10 μg/ml.

c) Sera Samples

Samples of known status have been used for the development of thisindirect ELISA. For Type I PRRSV, a panel of serum samples from 34 pigsthat had been previously vaccinated with Porcilis® PRRS. For Type IIPRRSV, serial sera samples were obtained from 20 pigs that had beenvaccinated with Ingelvac® PRRS MLV. In addition to PRRSV positivesamples, 127 PRRSV negative samples from negative control animals wereused for validation of the indirect ELISA. These PRRS negative sampleswere collected from different locations over Switzerland (Bazenheid,Basel, Zürich [ShZh, Zh]) in 2012. All of these samples were alsoassayed in the Idexx PRRS ELISA.

d) Details of Indirect ELISA

(i) Coating of ELISA plates: A high binding plate (Greiner Bio-One762071-CED) was coated with 100 μl of antigen solution per well.Depending of the antigen used for the coating, the final concentrationwas 1 μg/ml for GP4_LV or 10 μg/ml for GP4_VR_2332_4. The coating bufferin which the antigen was diluted in was PBS 13 1× with a pH 9.6. Theincubation of the antigen took place at 4° C. without shaking overnight.

(ii) Blocking: After antigen incubation overnight, the plate was washedfour times with 300 μl of Washing Buffer (0.5% Tween-80 [Sigma P8074-500ml] in PBS 13 1×) using Tecan Hydroflex washer. Next, 200 μl of BlockingBuffer (StabilCoat Immunoassay Stabilizer [SurModics Prod.Nr. SC01-100])were added per well. The plate was incubated for 2 hours at roomtemperature without shaking. After incubation period, the BlockingBuffer was drained and the plate was dried in a 37° C. incubator for 2hours.

(iii) Dilution of sera samples: Before testing, samples were diluted1:50. The dilution took place in Sample diluent buffer (0.2% casein inStabilCoat).

(iv) Capture: To each well on the plate, 100 μl of the 1:50 dilution ofthe serum sample was added. The incubation of the samples took place atroom temperature for one hour without shaking. After the incubationperiod, the plate was washed four times with 300 μl of Washing Buffer(0.05% Tween-80 [Sigma P8074-500 ml] in PBS 13 1') using Tecan Hydroflexwasher.

(v) Detection: After washing the plate, 100 μl of anti Pig IgG (Fc) HRP(20 ng/ml Conjugate Diluent) were added per well. Incubation took placeat room temperature for 1 hour without shaking. Next, the plate waswashed four times with 300 μl of Washing Buffer (0.05% Tween-80 in PBS13 1×) using Tecan Hydroflex washer. After washing, 100 μl of TMBsubstrate (SurModics TTMB-1000-01) were added per well. The plate wasthen incubated at room temperature for 15 minutes without shaking. Forstopping the reaction, 100 μl of Stop buffer solution (0.5M H₂SO₄ [Fluka38294]) were added per well. The absorbance of the coloured reaction wasmeasured using Tecan Sunrise (Measurement wavelength: 450 nm; Referencewavelengths: 620 nm; Read mode: normal).

e) Determining Cut-Off Values

To set negative/positive cut off values, both the mean and the standarddeviation of the negative samples only (Bazenheid, Basel, Zürich [ShZh,Zh]) were calculated. Once both values were obtained, the standarddeviation was multiplied by a factor of 5; the product of thiscalculation was added to the negative mean previously calculated.

TABLE 1 Calculation of Cut-Off values Mean Negative Standard DeviationCut-Off (Neg. Samples of Negative Samples Mean + Std. Dev*5) GP4_LV40.07 0.02 0.17 GP4_VR_ 0.06 0.05 0.32 2332_4

f) Calculation of Sensitivity and Specificity

The sensitivity is calculated to know the probability that a test willgive a positive result when the disease is present and it can becalculated using the following formula and following the scheme above(TABLE 5):Sensitivity=(A/A+C)*100

On the other hand, the specificity of an assay will provide informationabout the probability that a test will give a negative result when thedisease is not present. It can be calculated using the formula below andfollowing the scheme above (TABLE 2):Specificity=(D/B+D)*100

TABLE 2 Scheme for calculating sensitivity and specificity Sample StatusPositive Negative Test Positive A B A + B Negative C D C + D A + C B + DTotal

g) Results

As indicated above animals that were vaccinated with EU or US type aswell as PRRSV negative animals were tested in order to calculate thespecificity and sensitivity of the assay (TABLES 3 and 4 and FIGS. 1 and2) to determine the specificity and sensitivity of the indirect ELISA, acut-off of 0.17 and 0.32 at herd level was calculated for GP4_LV4 andGP4_VR_2332_4, respectively. With the gathering of all the data, wedetermined that the specificity and sensitivity of GP4_LV4 was 99.41%and 55.80%, respectively, whereas for GP4_VR_2332_4, the specificity was98.91% and the sensitivity 45%. For the calculation of the diagnosticspecificity, the PRRS positive samples that were negative depending onthe GP4 peptide used were considered as negative.

For the ELISA, although the sensitivity is important, it is of higherinterest that the assay is specific, since the main objective of thisdetection method is to be able to correctly classify individuals asdisease-free and avoid false positives.

TABLE 3 Results of optimum ELISA conditions for GP4_LV4 (PRRSV Type I)TEST GP4_LV4 (EU) Total Positive Negative Type 1-Sera 34 19  15 Type2-Sera 20  0* 20 Negative PRRSV 152  1** 151 antibody serum samples

As one can take from TABLE 3, no cross detection between EU and US couldbe observed and only one false positive sample was observed by testingPRRSV negative antibody serum.

TABLE 4 Results of optimum ELISA conditions for GP4_VR_2332_(PRRSV TypeII) TEST GP4_VR_2332_4 (US) Total Positive Negative Type 1-Sera 34  0*34 Type 2-Sera 20 9 11 Negative PRRSV 151  2** 149 antibody serumsamples

TABLE 4 shows no cross detection between EU and US and only two falsepositive were observed by testing PRRSV negative serum.

EXAMPLES 2-8 AND 11

Examples 2-8 show the results of ELISA analysing blinded samples derivedfrom different pig herds or sera with known status (example 11). Theherein described ELISA can also be used for the assessment about theimmunological status of a herd related to the presence or absence ofneutralization antibodies directed against special epitopes of GP3 andGP4. Further on statements about a fresh or continuous infectionsituation in a given herd situation can be drawn by consideration thetiter level in animals.

Result interpretation for examples 2-8 need additional informationalongside the discriminating ELISA e.g. information about the singleanimal, herd status, and the result of a screening ELISA is needed.

For examples 2-8 and 11 the experiments were done in the identicalmanner as described for Example 1 with the exceptions that the antigensrepresented combinations of GP3 and GP4 peptides. The used peptidesequences in examples 2-8 are indicated in the following:

Peptides Used:

Type I (EU) PRRSV GP3 LV 30AA54 (SEQ ID NO. 1):ICMPCSTSQA ARGRLEPGRS NMCRKGHDRC GP4 LV4 (SEQ ID NO. 2):DINCFRPHGV SAAQEKISFG KSSQCREA VGTP Type II (US) GP3 VR (SEQ ID NO. 3):VCPPCLTRQA ATEIYEPGRS LWCRIGYDRC EEDHDELGFM GP4 VR (SEQ ID NO. 4):DISCLRHRDS ASEAIRKIPQ CRTAIGTP

The additional peptide sequences used in example 11 are indicated in thefollowing:

Type I (EU) PRRSV GP3 (SEQ ID NO. 5): STSQAARQRL EPGRNMWCKI GHDRCEERGP3 (SEQ ID NO. 6): STSQAARQRL EPGRNMW GP4 (SEQ ID NO. 7):FRPHGVSAAQ EKISFGKSS Type II (US) GP3 (SEQ ID NO. 8):YEPGRSLWCR IGYDRCGEDD GP3 (SEQ ID NO. 9): IYEPGRSLWC RIGYDRCGED DHDELGP4 (SEQ ID NO. 10): HRDSASEAIR KIPQCRTAI

EXAMPLE 2: PRRS NEGATIVE HERD (NUCLEUS HERD)

Herd Status: Animal tested derived from a boar nucleus herd. Boars willnever be vaccinated against PRRSV and are closed meshed measured for thepresence of PRRSV.

Animal Status: Animals are predicted to be negative for PRRSV directedantibodies.

Screening ELISA is negative as well as the discrimination ELISA.

Test interpretation: Animals are negative for PRRSV.

In the following the results are summarized in TABLES 5 and 6.

TABLE 5 Discriminatory Reference ELISA Test Type I Type II Info Idexx X3(EU) (US) Animal Site Info Neg X X X Boars PRRSV negative Pos farmTitre* <0.3 <0.3 Nucleus herd

TABLE 6 Actual test results Information Discriminatory ELISA to animalsOD 450 nm-620 nm Sample and PRRS Type I Type II Reference Test No Status(EU) (US) Idexx X3 ELISA 12-2414 BOARS 0.026 0.068 Neg 12-2415 PRRS0.060 0.032 Neg 12-2416 negativer 0.051 0.069 Neg 12-2417 animals 0.0360.034 Neg 12-2418 0.065 0.092 Neg

EXAMPLE 3: PRRS NEGATIVE HERD

Herd Status: Unknown. However, boars will never be vaccinated againstPRRSV and are closed meshed measured for the presence of PRRSV.

Animal Status: Unknown

Screening ELISA is negative as well as the discrimination ELISA.

Test interpretation: Animals are negative for PRRSV.

In the following the results are summarized in TABLES 7 and 8.

TABLE 7 Discriminatory Reference ELISA Test Type I Type II Idexx X3 (EU)(US) Info Animal Site Info Neg X X X Boars PRRS negative Pos herd Titre*<0.3 <0.3

TABLE 8 Actual test results Information Discriminatory ELISA to animalsOD 450 nm-620 nm Sample and PRRS Type I Type II Reference Test No Status(EU) (US) Idexx X3 ELISA 13-1107 PRRS 0.109 0.119 Neg 13-1108 negativeherd 0.079 0.082 Neg 13-1109 0.029 0.049 Neg 13-1110 0.037 0.057 Neg13-1111 0.057 0.058 Neg

EXAMPLE 4: HERD WITH REPEATED PRRS INFECTION

Herd Status: Herd was positive tested in the past for the presence ofPRRSV EU directed antibodies over years. Boars are continuously infected(repeated infections) with PRRSV. Boars will never be vaccinated againstPRRSV and are closed meshed measured for the presence of PRRSV.

Animal Status: Unknown

Screening ELISA is positive but not the discrimination ELISA.

Test interpretation: Animals losing their capability to establish aneutralising humoral immune response in a continuous, repeated PRRSVinfection situation. However, these animals turned positive in ascreening ELISA not measuring neutralising antibodies. These animals,even tested positive in the screening ELISA do not have anymoreneutralising antibodies, indicating that a considerable proportion ofantibodies are not present anymore.

Even aware that boars demonstrate only minor clinical signs of a PRRSVinfection (e.g. fever) those animals may have a minor resistancecapability against a heterologous PRRSV challenge (infection with PRRSVType II in this assumed case, as animals are infected with EU typePRRSV).

In the following the results are summarized in TABLES 9 and 10.

TABLE 9 Discriminatory Reference ELISA Test Type I Type II Idexx X3 (EU)(US) Info Animal Site Info Neg X X Boars Farm with Pos X seroreactors,Titre* <0.3 <0.3 Continuous EU infected herd

TABLE 10 Corresponding ELISA Results Discriminatory ELISA Information toOD 450 nm-620 nm Sample animals and Type I Type II Reference Test NoPRRS Status (EU) (US) Idexx X3 ELISA 13-19 Farm with 0.082 0.131 Pos!13-22 seroreactors, 0.070 0.061 Pos! 13-24 Continuous EU 0.076 0.100Pos! 13-25 infected 0.043 0.072 Pos! 13-26 herd 0.038 0.087 Pos!

EXAMPLE 5: REPEATED EU INFECTED BOAR HERD, FRESHLY INFECTED WITH US

Herd Status: Herd was positive tested in the past for the presence ofPRRSV EU directed antibodies over years and turned recently positive forUS PRRSV as proved by positive US PRRSV PCR results. Boars arecontinuously infected (repeated infections) with PRRSV. Boars will neverbe vaccinated against PRRSV and are closed meshed measured for thepresence of PRRSV.

Animal Status: Unknown

Screening ELISA is positive and discrimination ELISA too.

Test interpretation: Animals losing their capability to establish aneutralising humoral immune response in a continuous, repeated PRRSVinfection situation against EU PRRSV. However, these animals turnedpositive in the discrimination ELISA for PRRSV Type II (US). Theseanimals tested positive in the screening ELISA.

Titre for US PRRSV are very high (>2 OD in average) indicating that afresh infection with US PRRSV occurred in the herd. This testinterpretation could be confirmed by PCR test results, demonstratingthat the infection turned out to be in the viraemic phase.

Animals may have a minor resistance capability against a heterologousPRRSV challenge (infection with PRRSV Type II in this assumed case, asanimals are infected with EU type PRRSV). Sows at gestation day 90,vaccinated with one of the MLVs but infected with a counter field PRRSVisolate would be the more profound sub species to prove the limitedprotection of the animals.

In the following the results are summarized in TABLES 11 and 12.

TABLE 11 Discriminatory Reference ELISA Test Type I Type II Idexx X3(EU) (US) Info Animal Site Info Neg X Boar Continuous EU Pos X Xinfected Titre* <0.3 >1 herd (minimal 3 years), US positive sinceJanuary 2013

TABLE 12 Corresponding ELISA Results Information Discriminatory ELISA toanimals OD 450 nm-620 nm Sample and PRRS Type I Type II Reference TestNo Status (EU) (US) Idexx X3 ELISA 12-2448 Continuous 0.140 0.381 Pos!12-2449 EU infected 0.040 0.052 Neg 12-2450 herd 0.040 0.061 Neg 12-2451(minimal 3 0.045 0.057 Neg 12-2452 years), US 0.044 0.051 Neg 12-2453positive 0.043 0.055 Neg 12-2454 since 0.062 2.291 Pos! 12-2455 January0.095 2.055 Pos! 12-2456 2013 0.044 2.341 Pos! 12-2457 0.177 2.478 Pos!

EXAMPLE 6: MATERNAL INTERFERENCE OR BUILDING OF FIRST NEUTRALIZINGANTIBODIES

Herd Status: Piglets four weeks of age. Normally the age one week aftervaccination against PRRSV. It is unknown if the sows have beenvaccinated or not (based on the discrimination ELISA): It is predictedthat the sows have been vaccinated with Porcilis PRRSV (EU PRRSV).

Animal Status: Unknown

Screening ELISA is positive and discrimination ELISA, too.

Test interpretation: Animals are actually too young to establish ahumoral immune response against PRRSV. It is likely to assume thatmaternal interference (transfer of neutralising antibodies from sow topig via suckling). It might be also possible that some mAB derived fromthe piglet immune system. Infection with wild PRRSV is unlikely due tothe age of the piglets and the low titre measured in the discriminationELISA.

In the following the results are summarized in TABLES 13 and 14.

TABLE 13 Discriminatory Reference ELISA Test Type I Type II Idexx X3(EU) (US) Info Animal Site Info Neg X piglets of 4 weeks, unknown Pos XX fattening Titre* <1 <0.3

TABLE 14 Corresponding ELISA Results Information Discriminatory ELISA toanimals OD 450 nm-620 nm Sample and PRRS Type I Type II Reference TestNo Status (EU) (US) Idexx X3 ELISA 13-2721 unknown 0.682 0.098 Neg13-2722 0.596 0.250 Neg 13-2724 0.175 0.035 Pos! 13-2725 0.923 0.206Pos! 13-2727 0.079 0.059 Neg

EXAMPLE 7: INGELVAC VACCINATED HERD (US PRRSV POSITIVE)

Herd Status: Piglets are ten weeks of age. Normally, this is the timesix weeks after the vaccination against PRRSV normally took place. Pigsshould demonstrate an intensive humoral immune response and high titrelevel are expected.

Animal Status: Unknown

Screening ELISA is positive and discrimination ELISA too.

Test interpretation: Animals are six weeks after vaccination. Based onthe fact that only US PRRSV could be detected, with very high titres(average>1.0 OD), it is likely that these animals have been vaccinatedwith Ingelvac® PRRSV (US PRRSV).

In the following the results are summarized in TABLES 15 and 16.

TABLE 15 Discriminatory Reference ELISA Test Type I Type II Idexx X3(EU) (US) Info Animal Site Info Neg X fattening, 30 kg, unknown Pos X Xca. 10 weeks of age Titre* <0.3 >1

TABLE 16 Corresponding ELISA Results Information to animalsDiscriminatory ELISA and OD 450 nm-620 nm Sample PRRS Type I Type IIReference Test No Status (EU) (US) Idexx X3 ELISA 13-2738 unknown, 0.0240.126 Neg 13-2743 30 kg 0.051 0.498 Pos! 13-2744 0.038 0.205 Pos!13-2748 0.031 0.756 Pos! 13-2749 0.033 0.691 Pos! 13-2753 0.055 2.894Pos! 13-2754 0.028 0.757 Neg 13-2755 0.030 0.177 Neg 13-2756 0.042 0.297Pos! 13-2757 0.085 0.405 Neg 13-2761 0.033 2.703 Pos! 13-2762 0.0682.545 Pos! 13-2763 0.023 0.395 Pos! 13-2764 0.039 0.193 Neg 13-27650.063 2.748 Pos! 13-2767 0.047 2.379 Neg 13-2768 0.113 2.788 Pos!13-2772 0.066 1.031 Pos! 13-2777 0.107 2.769 Pos! 13-2778 0.070 2.783Pos!

EXAMPLE 8: PIG HERD INFECTED WITH TYPE I AND II (EU AND US) OF PRRSV

Herd Status: Herd was positive tested in the past for the presence ofPRRSV EU directed antibodies over years and turned recently positive forUS PRRSV. Boars are continuously infected (repeated infections) withPRRSV. Boars will never be vaccinated against PRRSV and are closedmeshed measured for the presence of PRRSV.

Animal Status: Unknown

Screening ELISA is positive and discrimination ELISA for EU and US, too.

Test interpretation: Animals tested positive in the past for EU PRRSVand very recently for US PRRSV. These animals tested positive in thescreening ELISA. Titre for US PRRSV are very high (>1 OD in average)indicating that a fresh infection with US PRRSV occurred in the herd.Titre for EU PRRSV are low (<1 OD in average) indicating that an olderinfection with EU PRRSV occurred in the herd.

-   -   Old infection with EU PRRSV and fresh infection with US PRRSV

In the following the results are summarized in TABLES 17 and 18.

TABLE 17 Reference Discriminatory ELISA Test Type I Type II Idexx X3(EU) (US) Info Animal Site Info Neg Boars Pos X X X Titre* <1 >1

TABLE 18 Corresponding ELISA Results Information Discriminatory ELISA toanimals OD 450 nm-620 nm Sample and PRRS Type I Type II Reference TestNo Status (EU) (US) Idexx X3 ELISA Boar 1 Boars, 0.037 0.044 Neg Boar 20.031 0.029 Neg Boar 3 0.052 0.063 Neg Boar 4 0.039 0.038 Neg Boar 50.115 2.408 Pos! Boar 6 0.130 0.958 Pos! Boar 7 0.030 0.038 Neg Boar 80.035 0.063 Pos! Boar 9 0.071 1.544 Pos! Boar 10 0.267 2.584 Pos! Boar11 0.084 0.095 Neg Boar 12 0.125 2.614 Pos! Boar 13 0.035 0.423 Pos!Boar 14 2.310 0.964 Pos! Boar 15 0.440 1.539 Pos! Boar 16 0.455 1.156Pos! Boar 17 0.035 0.044 Pos! Boar 18 0.041 0.057 Pos! Boar 19 0.0290.031 Pos! Boar 20 0.037 0.036 Neg Boar 21 0.037 0.079 Pos! Boar 220.092 0.257 Pos! Boar 23 0.057 0.092 Pos! Boar 24 0.103 1.192 Pos! Boar25 0.040 0.053 Pos!

EXAMPLE 9: RESULTS OF ELISA ASSAYS ANALYZING SERUM SAMPLES OF KNOWN PRRSSTATUS

Discriminatory ELISA assays were performed on pig serum samples withknown PRRS Status. 32 sera derived of vaccinated pigs (Type I (EU)), 20sera derived of vaccinated pigs (Type II (US)) and 46 sera of PRRSVnegative pigs were analyzed on EU specific peptides (left column) and USspecific peptides (right column).

The results are depicted in TABLE 19.

The first number indicates amount of sample values over cutoff, rightnumber indicates the total number of sample analyzed per cohort. In the2 last rows the respective specificities and sensitivity is calculated.

TABLE 19 ELISA for ELISA for PRRSV Type 1 PRRSV Type 2 (EU) Peptides(US) Peptides GP3 and GP4 GP3 and GP4 Number of Number of PPRSV corectlytested corectly tested status of Type of PRRSV animals/Numberanimals/Number samples strain of tested animals of tested animalsPositive PRRSV Type 1 (EU) 20/32  7/32 Positive PRRSV Type 2 (US) 0/4619/20  Negative 0/46 2/46 Sensitivity  63% 95% Specificity 100% 97%

EXAMPLE 10: DIAGNOSTIC COMPOSITION (KIT)

A typical kit which can be used in the method according to the inventionincludes the components given in TABLE 20:

TABLE 20 Component Name Description Component 1 High binding 96-wellplate coated with PRRSV Type I/II Test Plate specific peptides asfollows: row 1, 3, 5, 7, 9, 11 coated with GP3/GP4 peptides specific forPRRSV Type I (EU) row 2, 4, 6, 8, 10 12 coated with GP3/GP4 peptidesspecific for PRRS Type II (US) Component 2 Buffer used for dilutingserum samples to be analyzed on Sample Diluent the Test Plate(Component 1) Component 3 Buffer used for rinsing unbound assaycomponents after Washing Fluid (20x) the sample incubation step andconjugate incubation step. Component 4 Horse radish peroxidase labelledanti-pig antibody used for Conjugate (30x) detection of antibodies boundto PRRSV Type I and Type II peptides. Component 5 Buffer used fordiluting the Conjugate (Component 4) Conjugate Diluent during theconjugate incubation step. Component 6 Serum of pig containing highantibodies titer specific for Positive Control EU PRRSV Type I (EU)Component 7 Serum of pig containing high antibodies titer specific forPositive Control US PRRSV Type II (US) Component 8 Serum of pigcontaining low antibodies titer specific for Weak Positive Control EUPRRSV Type I (EU) Component 9 Serum of pig containing low antibodiestiter specific for Weak Positive Control US PRRSV Type II (US) Component10 Serum of pig containing no antibody to the PRRS virus NegativeControl Component 11 Solution used as Enzymatic Substrate for HRPproducing a Substrate color reaction Component 12 Solution used forstopping and stabilizing the color Stop Solution development

EXAMPLE 11: PERFORMANCE OF COCKTAILS INCLUDING FURTHER GP3 AND GP 4PEPTIDES IN DIAGNOSTIC TESTS

For this example for each case 4 Type I (EU) positive, 4 Type II (US)positive and 4 PRRS negative samples were analyzed by indirect ELISA (asdescribed under example 1 in detail) using different cocktails ofantigens. The results are discussed below referring to FIGS. 3-6.

a) Cocktail of Type I (EU)-Specific GP3 and GP4 Peptides

In this assay peptides according to SEQ ID NO. 5 (GP3) and SEQ ID NO. 7(GP4) were used in combination. The results are shown in FIG. 3. One cansee that all 4 Type I positive samples showed a clear signal with highintensity while the remaining further samples were identified as clearlynegative.

b) Cocktail of Type I (EU)-Specific GP3 and GP4 Peptides

In this assay peptides according to SEQ ID NO. 6 (GP3) and SEQ ID NO. 2(GP4) were used in combination. The results are shown in FIG. 4. One cansee that all 4 Type I positive samples were identified positive with,however, signals of different intensity depending on the sample. Theremaining Type II (US) positive and PRRS negative samples showed onlyweak signals and could still be clearly discriminated as negative.

c) Cocktail of Type II (US)-Specific GP3 and GP4 Peptides

In this assay peptides according to SEQ ID NO. 8 (GP3) and SEQ ID NO. 10(GP4) were used in combination. The results are shown in FIG. 5. One cansee that all 4 Type II positive samples were identified positive with,however, signals of different intensity depending on the sample. Theremaining Type I (EU) positive and PRRS negative samples showed onlyweak signals and could still be clearly discriminated as negative.

d) Cocktail of Type II (US)-Specific GP3 and GP4 Peptides

In this assay peptides according to SEQ ID NO. 9 (GP3) and SEQ ID NO. 10(GP4) were used in combination. The results are shown in FIG. 6. One cansee that all 4 Type II positive samples were identified positive withsignals of higher intensity compared to example c. The remaining Type I(EU) positive and PRRS negative samples again showed only weak or nosignals and could still be clearly discriminated as negative.

The invention claimed is:
 1. A diagnostic composition for the detectionand classification of PRRSV-infections in swine herds, including atleast one antigen capable to bind a neutralizing antibody against theType I-virus possibly present in the animal wherein the at least oneantigen capable to bind a neutralizing antibody against the Type I-virusis a peptide of a PRRSV GP3 protein and is SEQ ID NO. 1 and at least oneantigen capable to bind a neutralizing antibody against the TypeII-virus possibly present in the animal and the necessary buffers andsolutions typically present in an ELISA-assay.
 2. The diagnosticcomposition of claim 1, wherein the at least one antigen capable to bindto neutralizing antibody against the Type II-virus is a peptide of aPRRSV GP3 protein and is SEQ ID NO.
 3. 3. The diagnostic composition ofclaim 1, wherein the at least one antigen capable to bind a neutralizingantibody against the Type I-virus possibly present in the animal and atleast one antigen capable to bind a neutralizing antibody against theType II-virus possibly present in the animal are coated on a plate. 4.The diagnostic composition of claim 1, wherein the at least one antigencapable to bind a neutralizing antibody against the Type I-viruspossibly present in the animal and at least one antigen capable to binda neutralizing antibody against the Type II-virus possibly present inthe animal are coupled to beads.